1. Field of the Invention
The present application is directed to a single cell preparation method, or a brain cell preparation that provides near-optimal conditions for the accurate quantitation of gene expression in the single cell or neuron. The present invention also provides for a method for studying the functional activity in the same single cell. Also, the method of the present invention relates to the collection of a single neuron, with its membrane and dendritic processes substantially intact and with a full set of mRNA transcripts.
2. Brief Description of the Related Art
With the remarkable explosion in gene expression xe2x80x9cchipxe2x80x9d technology in recent years it appears that it will soon be possible, perhaps routine, to measure the expression of hundreds or thousands of genes simultaneously, under a wide variety of physiological and pathological conditions. In the foreseeable future, this new technology will clearly overwhelm our capacity to analyze the functional implications of even a fraction of the gene expression patterns that will be found. Taking the central nervous system (CNS) as an example, eventually unraveling the complex linkages between multiple gene expression and CNS function will likely require studies of physiological properties in the same neuron in which multiple gene expression is also assessed.
In turn, such studies will depend on the availability of highly specialized preparations. An optimal CNS preparation for relating gene expression to function in the same neuron should seemingly have three major attributes: 1) the ability to perform extensive functional studies on the same single neuron that is subsequently collected for gene expression analysis; 2) the ability to collect the full set of mRNA transcripts from that same neuron; and 3) the ability to collect the neuron with most of its processes intact, in order to preserve subcellular and dendritic mRNA distribution. However, the main preparations commonly used in brain studies of gene expression, including assays in homogenized tissues (RNAse protection assays, Northerns), in situ hybridization, acutely dissociated neurons, or electrophysiological recording with extraction of cytoplasmic contents (e.g., in non-dissociated slices or cultures), do not provide optimal conditions for parallel measures of function and gene expression in the same neuron. In particular, none of these approaches routinely allows for the collection of the entire cell, including such specialized cells as neurons with their dendritic processes intact. Without a full set of mRNA transcripts, accurate estimates of specific mRNA content are difficult to obtain, and specific dendritically-targeted mRNAs are lost.
Gray et al. discloses a partially-dissociated hippocampal slice (e.g., the xe2x80x9czipper slicexe2x80x9d which gradually opensxe2x80x94or unzipsxe2x80x94along the cell body layers) in young guinea pigs to provide improved accessibility to neurons for patch clamp pipettes. However, Gray et al. does not disclose the method of the invention whereby a substantially intact single cell is isolated whereby substantially all of the mRNA is detected.
The invention brings together technology encompassing cutting edge instrumentation for electrophysiology, confocal laser scanning microscopy (CLSM), immunoautoradiography and histochemistry, real-time detection of PCR kinetics and new DNA xe2x80x9cchipxe2x80x9d technology (GeneChip Scanner and Analysis Suite), to optimize and extend the invention.
While there is clearly growing recognition of the value of single cell expression-function studies in the CNS, it seems less well recognized that the preparations that are most widely available for linking function to gene expression in single cells are significantly limited in their usefulness for these purposes. For example, the physiological recording methods in which cytoplasm is extracted through a pipette, do not yield the full complement of the cell""s mRNA nor do they allow correlations with topographic mRNA distribution. The proportion of total mRNA extracted varies so greatly from one cell to another that there have been few attempts to estimate the total amount of any mRNA species in a given neuron, or even to estimate relative or semi-quantitative levels. The observations based on this method to date have been generally limited to all-or-none types of findings, regarding, for example, whether or not a gene is expressed in a given cell, or in some cases, whether its ratio of expression to other genes (e.g., for different receptor subunits) is changed (e.g., Sudweeks and Twyman, 1995). In contrast, semi-quantitative or quantitative estimates of the absolute amount of expression of a gene in a cell generally requires collecting the entire complement of a neuron""s mRNA transcripts. This seems particularly critical for studies on function-expression correlation in the same cells since the physiological/pathological properties of a cell (e.g., density of channels or receptors, developmental stage, biochemical phenotype or pathological change) may well reflect total level of a gene""s expression rather than the ratio to another gene""s expression, which might also be altered.
Values obtained with the ratio normalizing approach required when fractional cellular contents are extracted, can be substantially affected by the additional error contributed by variability in the xe2x80x9ccontrolxe2x80x9d message to which the target message is normalized, by different efficiencies (e.g., in PCR) between the two messages, or by the very common occurrence in which the normalizing message is also up- or down-regulated in tandem, either by the conditions under investigation or by other conditions of the cell (e.g., size, metabolic activity) that affect both messages. The latter may sometimes provide a control for non-specific effects, but in more cases is likely to wash out the absolute value of the target gene signal with which the investigated function may be correlated. Further, it is becoming clear that differential targeting and distribution of mRNAs within the cell (e.g., dendrites or soma) play critical roles in the CNS (Steward et al., 1998; Kuhl and Shehel, 1998). The collection of mRNA by cytoplasmic extraction loses this differential distribution.
Thus, without the ability to obtain the full complement of mRNA with its topographic distribution intact, it will clearly be difficult to estimate the total amount of a mRNA species in a neuron. In turn, this will make it extremely difficult, in most cases, to draw quantitative conclusions about the relations between gene expression and physiological function in individual cells. It should be noted that although the term xe2x80x9cgene expressionxe2x80x9d is used here as a short hand somewhat interchangeably with mRNA content, this is only for purposes of simplicity and it is well recognized that the two are not necessarily equivalent. In the present application, we focus only on the quantitation of total mRNA transcripts, but controls are of course required in many types of experiment before concluding that mRNA content directly reflects expression.
Conventional Methods for mRNA Expression Measurement in the Brain
The main available preparations are: a) Homogenized Tissues: Neuronal and glial heterogeneity generally prevents the accurate assessment of gene/mRNA expression relative to specific cell types or functions, even if small regions of brain are dissected, homogenized, and analyzed by conventional methods (RPAs, Northerns) b) In Situ Hybridization allows visualization of topographic mRNA expression in single cells, but because the tissue is fixed, does not usually permit functional measures (electrophysiology, optical imaging) from the same cells or collection of the mRNA pool for subsequent amplification; also, quantitative analysis (grain counting) is performed by sampling on one section, lending some error to the estimate of total mRNA; c) Acute Dissociation of brain neurons disrupts the membrane and amputates processes. It is not suitable for use with aged or even mature adult neurons as these are often highly traumatized by the procedure (Thibault et al., 1995a); in addition the loss of dendritic processes precludes studies of topographic differences in gene expression or collection of total mRNA; and d) Electrophysiological Whole Cell Recording (e.g., slices or culture) is compatible with many types of functional studies but generally collects mRNA by aspiration of cytoplasmic content, which as noted, is highly variable from cell to cell and prevents reliable collection of the full set of mRNA transcripts or separate study of somal and dendritic compartments. Further, the Whole-cell method dialyzes the cell""s interior which can dilute substances that modify physiological function.
Thus, despite the rapidly developing sophistication in measuring multiple gene expression, the currently available preparations are not well suited for careful physiological-expression correlation studies in the same neurons or even for collecting total mRNA in a single neuron. However, as noted above, the partially-dissociated, or xe2x80x9czipperxe2x80x9d, brain slice (Gray et al., 1990), appears to have the potential to be a nearly ideal preparation for such studies in brain cells of mammals of any age range (Thibault et. al., 1995a; Thibault and Landfield, 1996;Chen et al., 1998 and Preliminary Data).
Function and Expression: Statistical Value of Single Cell Correlations
It is becoming increasingly clear that there is considerable variability in the expression responses of different neuron types in the same brain region and even among different neurons of the same type. For example, it has been found that different neuron types and/or neurons of the same general phenotype can exhibit very different quantitative or topographical (dendrites vs. soma) patterns of distribution of the same mRNA species. One recent major study concluded that there are no general rules for mRNA localization that apply to all neuron types nor are there neuron-type-specific mechanisms that invariably regulate mRNA distribution (Paradies and Steward, 1997).
Thus, testing a hypothesis that some aspect of gene expression is directly linked to a specific function will in many cases require correlational analyses of the degrees of association across these highly variable individual cellular patterns. Statistically, individual-sample correlation of course provides a more rigorous test than co-variance among group means, since the degrees of freedom (df) across which a possible correlation can vary in a study of, say, 20 neurons in which both a physiological process and mRNA content are measured in response to a treatment in each neuron, would be 19 [df=n-1(20-1)]. However, if functional and expression values are obtained separately in different neuron groups (e.g., one set of neurons for recording, and one for mRNA), then the physiology-mRNA correlation can only vary around the number of experimental conditions (group means) and the associated degrees of freedom (e.g., treatment or no treatment). Therefore, the many more df""s generally found in an individual sample correlation study allow for clear statistical inferences and probabilistic statements on the amount of variance in one variable that is accounted for by variance in the other. This is not possible for associations involving few df""s (e.g., typically across group means) which are consequently more susceptible to chance associations.
A more general problem in this regard is that most major treatments or conditions (e.g., aging, seizures, intense synaptic stimulation, lesions, drugs, hormones, neurodegenerative disease, developmental stages, etc.) presumably activate a large number of genes. As the new microarray techniques for simultaneously assessing thousands of genes increasingly come on line, it will become extremely difficult to determine which if the many observed changes in expression are relevant to function without careful same-cell observations of both function and expression. Thus, the application of function-gene expression correlations in single cells (e.g., with large multiple regression correlation matrices and appropriate controls for performing many comparisons) may become one of the key first steps in attempting to interpret widespread gene activation in relation to function and in dealing with the vast quantities of data that the field is on the threshold of obtaining.
Prior Studies on Electrophysiology and Single Cell RT-PCR
The vast majority of previous studies on electrophysiological recording and RT-PCR in the same single neurons have, as noted, addressed all-or-none or message ratio questions, usually related to whether or not a cell expresses a specific mRNA, and, if so, whether or not it also manifests a particular physiological property. For example, the GABAA receptor is thought to be composed of 5 subunits, but there are almost 20 known subunits and variants that can form the GABAA receptor. In transfected cells, different combinations can influence affinity, pharmacological modulation, channel conductance and single channel kinetics (e.g., Porter et al., 1992). However the actual subunit combinations that occur in vivo are not known. Consequently, many single cell recording-PCR studies have been used to determine which subunits are expressed in which cells, and how these combinations affect function (Sudweeks and Twyman, 1996).
Although several prior studies have attempted semi-quantitative analyses, a recent report (Tkatch et al., 1998) indicated a quantitative correlation between an electrophysiological function (K+ channel A-currents) and a measure of gene expression for a related subunit (mRNA for the Kv4.2 K+ channel) in individual brain neurons. However, that study was performed in acutely dissociated basal ganglia neurons and therefore could not collect total mRNA. In addition, a few studies in peripheral or invertebrate neurons have also quantitatively correlated physiological function and gene expression (e.g. Baro et al., 1997). But no one has accomplished the isolation of a single cell neuron having complexes processes as in an embodiment of the present invention.
There is clearly an overall paucity of CNS single cell studies of electrophysiological function-gene expression correlations, very likely because of the limited availability of preparations compatible with quantitative analyses.
The Partially-Dissociated (xe2x80x9cZipperxe2x80x9d) Slice. The partially-dissociated slice preparation (often termed the xe2x80x9czipper slicexe2x80x9d for its tendency to gradually open, or unzip, along the cell body layers), was originally developed by Gray, Johnston and colleagues (Gray et al., 1990) in young guinea pigs. The partial dissociation (unzipping) procedure involves mild enzymatic exposure to proteolytic enzymes and gentle xe2x80x9cshakingxe2x80x9d (FIG. 1). It provided unparalleled access to brain neurons for small patch pipettes and therefore yielded the high quality recordings needed for single channel analyses, with very little disturbance of cell structure. This adaptation incorporated somewhat shorter and lower enzyme exposure and more gradual xe2x80x9cunzippingxe2x80x9d (Thibault et al., 1995a), and required several months to optimize. Of particular importance was that the yield of high resistance ( greater than 20 Gxcexa9) recordings from healthy neurons was equivalent from young adult, mid-aged and aged rat slices (Thibault and Landfield, 1996). Thus, with this preparation, we were able to carry out the first single channel analyses in brain neurons of aged mammals, and found an aging-related increase in the estimated membrane density of available L-type voltage sensitive Ca2+ channels (VSCC) (Thibault and Landfield, 1996) (FIGS. 2, 3).
Although each preparation has advantages for certain kinds of electrophysiological studies, it was noted above that neither the acutely dissociated cell preparation nor the non-dissociated slice or culture preparation permits consistent collection of the entire mRNA complement in a single cell. Aspiration of cytoplasmic contents through a whole cell patch pipette, in either of these preparations yields a varying and unknown fraction of the mRNA content from cell to cell (e.g., significant mRNA is likely trapped by organelles or the collapsing cell structure, and most dendritic mRNA is probably trapped in collapsing dendrites).
Moreover, most current electrophysiological preparations are extremely limited in their usefulness for applications to mature adult, much less aged, animals. Cell cultures generally utilize embryonic or postnatal neurons and acute dissociation techniques are usually focused on juvenile animals. In fact, we found that acute dissociation was so traumatic for aged rat brain cells that almost none survived the dissociation procedure (Thibault et al., 1995a). Even most non-dissociated slice studies are performed on juvenile or very young adult animals (cf. reviews, Thibault et al., 1995a; 1998a).
In the zipper slice however, the neuron that is being recorded with a cell-attached pipette can be easily and gently extracted from the slice, with nearly all of its processes intact simply by gradually withdrawing the pipette while still maintaining negative pressure on the cell body. The entire long apical dendritic tree slides readily out of the slice still attached to the non-disrupted cell body. Most of the basilar dendrites also appear to be intact (FIG. 4). Aged animal neurons are extracted as readily and as non-traumatically as are young. Thus, not only is the zipper slice particularly suitable for large scale single channel recording studies, but it appears to be a neurobiological preparation that can provide a fully intact neuron with its morphological structure preserved, for analysis of gene expression. Although this preparation has usually been used with hippocampus, there appears to be no reason why the preparation would not work from essentially any brain region, or with any tissue of an animal in an animal of any age.
Another method that can yield semi-quantitative estimates of mRNA expression in a single neuron that is largely topographically intact is in situ hybridization. However, even this approach generally yields only a sample of total mRNA (i.e., on the section through the cell) and can only be related to functional measures of the same cell with difficulty.
Limitations of the Zipper Slice
Although the zipper slice is presently extremely well suited for ion channel and imaging studies (cf. below), its main limitation appears to be its sub-optimal suitability for synaptic studies. The weakening of synaptic and tissue connections that makes it so ideally suited for cell extraction also results in variable synaptic connections, and therefore the zipper slice yields inconsistent results in synaptic studies. However, the present invention overcomes this limitation and greatly expands the range of functional studies for which the zipper slice is highly valuable.
A method is described for isolating a single cell from its organ tissue, usually a neuron from neural tissue, while causing minimal disruption of the cell""s processes and membrane. The method of the invention comprises extracting that cell from the tissue mass, washing and transferring the cell, and then collecting the entire cell. In the case of neurons, the processes are substantially intact.
The method of the invention further comprises determining the presence or amount of nucleic acid that is present in the extracted single cell. The inventive method comprises collecting the entire cell into a small pipette or tube filled with solutions and substances that facilitate the detection by amplification or hybridization of the messenger ribonucleic acid (mRNA) transcripts. The cell membrane is then disrupted and methods for amplification and art-accepted measurement techniques for measurement of small quantities of mRNA or deoxyribonucleic acid (DNA) are applied.
In a preferred example, a brain slice from an experimental animal is placed in a perfusion chamber and kept alive by oxygenation and artificial cerebrospinal fluid (ACF). The slice is then subjected to mild enzymatic concentrations and is nicked (cut) in a way such that gradual dissociation of the slice occurs along the cell body layers. Gentle vibration enhances this dissociation.
When the dissociation has proceeded sufficiently to expose cell bodies, one of the cells is xe2x80x9cpatchedxe2x80x9d onto a glass patch pipette (electrode) using standard patch clamp recording procedures. These procedures involve suction (negative pressure) in the pipette, and result in a tightly formed seal between the cell membrane and the tip of the pipette. This tight seal facilitates low noise amplification and recording of the electrical activity of the cell.
After the recording or other physiological monitoring session is completed, the cell is then pulled out of the tissue slice with most of its processes intact by maintaining the suction pressure of the pipette (electrode) and withdrawing the pipette by use of its micromanipulator controls. The cells remain attached to the pipette tip and readily slide out of the tissue mass with this method. The nerve cell on the tip of the pipette is optionally washed in clean ACF to remove extraneous mRNA and it is then transferred to a larger collection or harvesting pipette filled with an appropriate reverse transcriptase (RT) solution. The membrane is then disrupted osmotically to allow the RT process to begin. The contents of the cell are then transferred again to a tube containing an appropriate solution for amplification, for example, by polymerase chain reaction (PCR). Other methods for amplifying mRNA or DNA would be equivalent to RT-PCR for purposes of the invention.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.